Stabilized Pharmaceutical Composition Containing Pemetrexed Or Pharmaceutically Acceptable Salt Thereof

ABSTRACT

The present invention relates to a pharmaceutical composition with enhanced stability, containing pemetrexed or a salt thereof, and a preparation method therefor. The present invention provides an injection preparation in liquid form containing pemetrexed, capable of ensuring sufficient stability during circulation and storage by selection of an optimum material and setting of an optimum concentration range in order to secure the stability of pemetrexed. The present invention can provide a pemetrexed preparation which is readily commercially prepared, can prevent microbial contamination occurring during lyophilization or reconstitution, and has enhanced convenience of administration and stability.

TECHNICAL FIELD

The present invention relates to a stabilized pharmaceutical compositioncontaining pemetrexed or a pharmaceutically acceptable salt thereof, andto a method for producing the same.

BACKGROUND ART

Pemetrexed or a salt thereof (for example, disodium salt) exhibits ananti-cancer effect by inhibiting the activities of metabolites that areinvolved in a metabolic pathway of folate in various kinds of cancersincluding mesothelioma and non-small cell lung cancer. As amultitargeted antifolate, pemetrexed is known to target thymidylatesynthase (TS) and dihydrofolate reductase (DHFR) when it is activatedinto polyglutamate derivatives by folylpolyglutamate synthetase (FPGS),after it is introduced to the cells via reduced folate carrier (RFC),which is a major intracellular transport channel for folates(Chattopadhyay, S. et al. 2007. Pemetrexed: Biochemical and cellularpharmacology, mechanisms, and clinical applications. Mol. Cancer Ther.6, 404-417).

Chemical name of pemetrexed isN-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-gluatamicacid with the following chemical structure (see, U.S. Pat. No.5,344,932).

Presently, pemetrexed is developed under the trade name of ALIMTA®, andin South Korea, it has been marketed since 2007 and used in combinationwith cisplatin for a patient having inoperable malignantpleuralmesothelioma, who has never received chemotherapy, or it is usedas a monotherapy agent for locally advanced breast cancer and non-smallcell lung cancer after previously carrying out chemotherapy. ALIMTA® issold in the market in the form of a lyophilized formulation which needsto be reconstituted prior to administration, i.e., as a formulation inlyophilized powder form (100 mg or 500 mg) that is required to bereconstituted with 0.9% sodium chloride solution and diluted (finalconcentration at 0.25 mg/ml) finally with 0.9% sodium chloride solutionfor administration to a patient.

Due to the instability of a pharmaceutical in an aqueous solution phase,an injection preparation of the lyophilized formulation is firstlyprepared in lyophilized powder form and used after reconstituting itwith physiological saline or water for injection prior to administrationto a patient. However, a process for the reconstitution is cumbersome inthat the necessary amount needs to be weighed and added to a vial for alyophilized product, and there is a risk of having microbialcontamination during the reconstitution process. Furthermore, there isalso a limitation that it needs to be used within a pre-determined timeafter the reconstitution. Furthermore, because many hours are requireddue to a long drying cycle of the lyophilization process, thelyophilized formulation has a problem in that it has high productioncost and complicated production process. Accordingly, when the economicfeasibility of production, user convenience, and the like areconsidered, there has been a need for a ready-to-use liquid compositionhaving ensured stability.

In many cases, the problem of a liquid preparation lies in theinstability during storage period. Due to the instability, manyinjection preparations are used in lyophilized preparation form which isused after dissolution just before injection. Meanwhile, pemetrexed is arepresentative example of pharmaceuticals which undergo rapid oxidationin an aqueous solution to yield various kinds of impurities.

As a method of using an anti-oxidant to enhance the stability of aliquid preparation, a liquid pemetrexed preparation containing ananti-oxidant selected from the group consisting of monothioglycerol,L-cysteine, and thioglycolic acid is known in U.S. Pat. No. 6,686,365(Korean Patent Application Publication No. 2002-0081293) as a stablepemetrexed solution preparation which contains a therapeuticallyeffective amount of pemetrexed, an effective amount of an anti-oxidant,and a pharmaceutically acceptable excipient. In the examples of theaforementioned patent, a composition using, as an anti-oxidant, 2.4mg/mL monothioglycerol, 0.03% L-cysteine, and thioglycolic acid isdescribed. However, a stability improvement level based on a specificexperimental example like safety evaluation is not given therein.Furthermore, optimum type and optimum concentration of the anti-oxidantare not selected by specific examples. Still furthermore, when theinventors of the present invention evaluated the safety by reproducingthe examples of the aforementioned patent, results like a change inproperties like discoloration, an increase in impurities, or the likeare exhibited within 4 weeks of storage after carrying out a safety testunder accelerated conditions (40° C., 75% RH) in all the examples. Assuch, the inventors of the present invention acknowledged a need fordeveloping a more specific and advanced technique for allowing supply ofa liquid formulation containing pemetrexed, which has sufficientstability during circulation and storage, and thus they started thestudy on the present invention.

Furthermore, in Korean Patent Registration No. 1260636, a preparationhaving enhanced stability by using acetyl cysteine as an anti-oxidantand citric acid as a buffer agent with pemetrexed is disclosed, and inKorean Patent Application Publication No. 2013-0122065, an attempt ismade to provide a composition having enhanced stability by containing atleast one stabilizing agent selected from sodium sulfide and sodiumsulfite as an anti-oxidant. As a result of evaluating the stability of aliquid type which has been prepared according to the examples of theabove patent, sufficient stability was not exhibited due to a change inproperties like discoloration, an increase in impurities, or the likewithin 4 weeks at 60° C., and thus it was not recognized as an easilyreproducible technique. Furthermore, in the specification, the result ofobserving the safety only for 4 weeks at 60° C. is described, and thereis absolutely no mention of the stability for a period over 4 weeks.Accordingly, the inventors of the present invention intended to providea technique that is easily reproducible and enables the stability for 3months or longer, and more preferably for a sufficient time period like6 months at 60° C. or higher.

According to the process of searching various anti-oxidants, theinventors of the present invention found that, among the anti-oxidantsthat can be used in the pertinent field, monothioglycerol can remarkablyenhance the stability of pemetrexed. It is generally known that, when ananti-oxidant is used to suppress the decomposition of apharmacologically active substance caused by oxidation and an increasein impurities, the safety is enhanced in proportion to the additionamount of an anti-oxidant if it is contained at certain level or higher.However, unlike the descriptions given in prior patent inventions, theinventors of the present invention found that there is a negative effectof monothioglycerol on the stability of pemetrexed when monothioglycerolis present at concentration of 1.50 mg/mL or higher. As such, byselecting an optimum material and also setting an optimum concentrationfor ensuring the stability of pemetrexed, the inventors of the presentinvention succeeded in providing a liquid injection preparationcontaining pemetrexed which allows securing of sufficient stabilityduring circulation and storage and also is stable for 3 months orlonger, and more preferably 6 months or longer at 60° C. Consequently,it becomes possible to supply a liquid pemetrexed preparation of whichstability can be maintained for a long period of time at hightemperatures.

It is also required to minimize a contact with oxygen to secure thestability of a pharmaceutical which is decomposed according to anoxidation mechanism. As such, to reduce the decomposition of pemetrexedresulting from a contact with oxygen, the amount of dissolved oxygenpresent in solution and oxygen content in the headspace of a vial needto be controlled. As a result of various trials, the inventors of thepresent invention confirmed that sufficient stability can be obtainedwhen the oxygen content of a liquid composition provided by the presentinvention is controlled to about 1.5% or less, and preferably 1.0% orless in the headspace of a vial. In this regard, a method of fillinginert gas (for example, nitrogen and argon) as a separator in theheadspace of a vial is employed.

As a result, according to selection of an optimum anti-oxidant andsetting of an optimum concentration of an anti-oxidant and standardoxygen concentration in the headspace of a vial, it becomes possible toprovide a liquid formulation containing pemetrexed which has remarkablestability when compared to a known technique and can be stably providedas a commercially available product.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In consideration of the problems that are described above or the like,the inventors of the present invention conducted various studies forimproving the stability of a pharmaceutical composition containingpemetrexed or a salt thereof in the form of a solution for injection,and according to the processes of screening various anti-oxidants, theinventors found that monothioglycerol has an excellent anti-oxidanteffect while it uniquely shows a decreased anti-oxidant effect at theconcentration of 1.50 mg/mL or higher. By also setting the standard fordissolved oxygen in the composition constituting the present inventionand oxygen content in the headspace of a vial, the stability is furtherenhanced.

Means for Solving Problem

To secure the safety of pemetrexed in a solution state, the presentinvention provides a liquid preparation for injection containingpemetrexed, which is prepared by dissolving pemetrexed or apharmaceutically acceptable salt thereof in an aqueous solvent, havingmonothioglycerol at concentration of 0.50 to 1.25 mg/mL as ananti-oxidant, and, after filling with a pharmaceutical solution,controlling oxygen in the headspace of the vial to 1.5% or less, andpreferably 1.0% or less.

According to the present invention, the concentration of pemetrexed or apharmaceutically acceptable salt thereof is about 1 to 100 mg/mL,preferably 20 to 100 mg/mL, and most preferably 25 mg/mL.

According to U.S. Pat. No. 6,686,365 (Korean Patent ApplicationPublication No. 2002-0081293), use of monothioglycerol as ananti-oxidant is suggested as a way of ensuring the stability ofpemetrexed in the form of a solution and a liquid preparation containing2.4 mg/mL of monothioglycerol is simply disclosed in the examples.Namely, specific experimental results relating to the stability are notprovided. On the contrary, the absence of anti-oxidant effect at theabove concentration is confirmed by the process of the presentinvention. It is also disclosed that the anti-oxidant effect isexhibited when monothioglycerol is contained at certain concentration orhigher.

Based on the experiments using various concentration ranges over a longperiod of time, the inventors of the present invention confirmed thatthe optimum anti-oxidant effect is shown when monothioglycerol iscontained at 1.25 mg/mL or lower, and if monothioglycerol is containedat concentration higher than that, the anti-oxidant effect issignificantly reduced. Furthermore, because lower side effect of aliquid preparation on a human body and lower production cost can beobtained by reducing the content of an anti-oxidant, it is found thatmonothioglycerol needs to be contained at a concentration of at least0.50 mg/mL in order to maintain the aforementioned advantages whilemaintaining an excellent anti-oxidant effect. Accordingly, it is foundthat the stability of pemetrexed is significantly enhanced in a liquidcomposition which contains monothioglycerol at a concentration of 0.5mg/mL to 1.25 mg/mL, preferably 0.50 to 1.00 mg/mL, and most preferably0.50 to 0.80 mg/mL.

Furthermore, the stability of pemetrexed is significantly enhanced in aliquid composition in which pemetrexed or a pharmaceutically acceptablesalt thereof as an active ingredient and monothioglycerol as ananti-oxidant are contained at a ratio of about 20:1 to 50:1, andpreferably about 25:1 to 50:1.

As a way of minimizing the decomposition of pemetrexed caused by acontact with oxygen, by setting the dissolved oxygen amount in solutionat 0.5 ppm or lower and setting the oxygen content in the headspace ofthe vial at about 1.5% or lower, and preferably about 1.0% or lower, aliquid type injection preparation containing pemetrexed with sufficientstability during circulation and storage can be provided.

According to an evaluation based on product quality managementstandards, the preparation provided by the present invention isconfirmed to have the stability that is equivalent to the stability ofan existing injection formulation in solid form, in terms of an increasein decomposition products including discoloration for 3 months orlonger, and preferably for 6 months at severe (60° C.) stabilityconditions.

The pemetrexed preparation of the present invention contains pemetrexedor a pharmaceutically acceptable salt thereof as an active ingredient.

As described herein, the expression “pemetrexed” indicates a5-substituted pyrrolo[2,3-d]pyrimidine compound specifically representedby the following chemical formula 1, and it means a multitargetedantifolate which exhibits an anti-cancer effect in various kinds ofcancers including non-small cell lung cancer and malignantpleuralmesothelioma.

As described herein, the expression “pharmaceutically acceptable salt”means a salt that is produced by a method commonly used in the pertinentart field. Specifically, the pharmaceutically acceptable salt includessalts that are derived from a pharmaceutically acceptable inorganicacid, organic acid, or base, but not limited thereto. In particular, thepharmaceutically acceptable salt of pemetrexed can be a disodium salt ofpemetrexed which is currently commercially available, but not limitedthereto.

As described herein, the expression “pemetrexed or a pharmaceuticallyacceptable salt thereof” has a concept of encompassing a hydrate ofpemetrexed or a pharmaceutically acceptable salt thereof, and hydratesin any form, for example, 2.5 hydrate, 7 hydrate or the like areincluded, but not limited thereto.

According to the present invention, the pemetrexed preparation maycontain a pharmaceutically acceptable carrier and a pH controllingagent.

According to the present invention, the pemetrexed preparation ispreferably a liquid preparation that is storable in a solution state,and it is more preferably a ready-to-use liquid preparation forinjection contained in a sealed container.

According to the present invention, when the pemetrexed preparation is aliquid preparation for injection, the pharmaceutically acceptablecarrier is water for injection.

For producing the preparation of the present invention, inert gas suchas nitrogen or argon can be used to remove, by deaeration, dissolvedoxygen in a solution for injection. Methods for the deaeration can beused in combination, and vacuum deaeration and N₂ bubbling deaeration,vacuum deaeration and membrane deaeration, or vacuum deaeration andcatalyst resin deaeration or the like can be used in combination, forexample. Furthermore, the deaeration can be carried out one or moretimes.

According to the present invention, pH of the liquid pemetrexedpreparation for injection is preferably about 6.0 to 8.0, and morepreferably about 7.2 to 7.8. pH of the solution can be adjusted by usingan acid like hydrochloric acid or a base like sodium hydroxide.

The pemetrexed preparation of the present invention may not contain anadditive other than monothioglycerol described above. However, it mayadditionally contain a pharmaceutically acceptable excipient. As for thepharmaceutically acceptable excipient, a well-known additive such aslactose, dextrose, cyclodextrin and derivatives thereof, sucrose,glycerol, or sodium carbonate can be mentioned. Preferred examples ofthe excipient include sodium chloride and mannitol, but not limitedthereto.

The stabilized pemetrexed-containing solution for injection of thepresent invention can be packaged in a suitable container that is knownin the pertinent art field. Examples of a suitable container include aglass vial, a glass bottle, a cartridge, a pre-filled syringe, andanything similar to them, and it is preferably a glass vial.

The preparation is dispensed by adding it in a container which has beenwashed and sterilized in advance and sealed with a Teflon stopper ofwhich surface is suitable for the container and inert to the aqueouspemetrexed solution. The stopper is attached by using a crimper.

Effect of the Invention

According to the present invention, a pemetrexed preparation which isreadily commercially prepared, can prevent microbial contaminationoccurring during lyophilization or reconstitution, and has enhancedconvenience of administration and stability can be provided.Specifically, based on selection of an optimum anti-oxidant, setting ofan optimum concentration of the anti-oxidant, and management of oxygenconcentration in the headspace of the vial for maximizing the stabilityof pemetrexed in a solution, the stability is significantly enhancedcompared to a publicly known technique. Finally, by using the presentinvention, a pemetrexed-containing injection preparation in liquid form,which has sufficient stability during circulation and storage and isstable for 3 months or longer, and preferably for 6 months at 60° C.,can be supplied.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is more specifically explained in view ofExamples. However, the Examples are provided for illustrative purposeonly to aid the understanding of the present invention, and it isevident that the scope of the present invention is not limited by thefollowing Examples.

EXAMPLES Examples 1 to 4 and Comparative Examples 1 and 2 Production ofPemetrexed-Containing Solution for Injection According to VariousAnti-Oxidant Concentrations

D-Mannitol (25 g) was dissolved in 1,000 mL of water for injection, andmonothioglycerol was added at the concentration of the following Table 1and completely dissolved therein. To the resultant, 30.206 g (25 g interms of pemetrexed) of pemetrexed disodium 2.5 hydrate were slowlyadded and stirred till to have transparency. The resulting solution wassubjected to sterile filtration by using a sterilized 0.22 μm membranefilter, and dissolved oxygen concentration in the solution afterfiltration was adjusted to about 0.5 ppm by nitrogen purging. pH of theall solutions prepared according to Examples 1 to 4 and Comparativeexamples 1 and 2 was measured to be 7.6. The solution obtained fromabove was filled in a vial, and after controlling the oxygenconcentration in the headspace of the vial using nitrogen, it was sealedwith a rubber stopper and capped with an aluminum cap to have each ofExamples 1 to 4, and Comparative examples 1 and 2.

TABLE 1 Concentration Concentration Dissolved Oxygen of main of anti-oxygen concentration component oxidant amount in headspace (mg/mL)Anti-oxidant (mg/mL) (ppm) of vial (%) Example 1 25 monothioglycerol0.50 0.5 or less 1.0 or less Example 2 25 monothioglycerol 0.75 0.5 orless 1.0 or less Example 3 25 monothioglycerol 1.00 0.5 or less 1.0 orless Example 4 25 monothioglycerol 1.25 0.5 or less 1.0 or lessComparative 25 monothioglycerol 1.50 0.5 or less 1.0 or less example 1Comparative 25 monothioglycerol 0.10 0.5 or less 1.0 or less example 2

Examples 3, 5 and 6, and Comparative Examples 3 and 4 Production ofPemetrexed-Containing Solution for Injection According to Various OxygenConcentrations in Headspace of Vial

According to various oxygen concentrations in the headspace of the vialas shown in the following Table 2, a pemetrexed-containing solution forinjection was produced in the same manner as Example 3 above.

TABLE 2 Concentration Concentration Dissolved Oxygen of main of oxygenconcentration component anti-oxidant amount in headspace (mg/mL)Anti-oxidant (mg/mL) (ppm) of vial (%) Example 5 25 monothioglycerol1.00 0.5 or less 0.5 Example 3 25 monothioglycerol 1.00 0.5 or less 1.0Example 6 25 monothioglycerol 1.00 0.5 or less 1.5 Comparative 25monothioglycerol 1.00 0.5 or less 2.0 example 3 Comparative 25monothioglycerol 1.00 0.5 or less 5.0 example 4

Comparative Examples 5 to 10 Production of Pemetrexed-ContainingSolution for Injection According to Type of Anti-Oxidant

According to the composition and content shown in the following Table 3,a pemetrexed-containing solution for injection was produced in the samemanner as Example 3 above. In Comparative example 5, the production wasmade by using a solution in which only deaerated water for injection isused as a solvent without adding an anti-oxidant.

TABLE 3 Concentration Concentration Dissolved Oxygen of main of anti-oxygen concentration component oxidant amount in headspace (mg/mL)Anti-oxidant (mg/mL) (ppm) of vial (%) Comparative 25 — — 0.5 or less1.0 or less example 5 Comparative 25 L-Cysteine 1.00 0.5 or less 1.0 orless example 6 Comparative 25 Thioglycolic 1.00 0.5 or less 1.0 or lessexample 7 acid Comparative 25 EDTA 1.00 0.5 or less 1.0 or less example8 Comparative 25 Sodium 1.00 0.5 or less 1.0 or less example 9 sulfiteComparative 25 Sodium 1.00 0.5 or less 1.0 or less example 10 sulfide

Stability Test

For the compositions which have been obtained from Examples 1 to 6according to the present invention, and also from Comparative examples 1to 10, 6-month stability test was carried out at accelerated conditions(40° C., 75% RH) and severe conditions (60° C.). For the stability test,the amount of impurities which remain in the aqueous solution wasanalyzed and measured according to the following method in addition toobservation of the characteristics of the aqueous solution.

Conditions for HPLC Liquid Chromatography of Impurities

-   -   Detector: UV-Visible spectrophotometer (measurement wavelength:        250 nm)    -   Column: Zorbax SB-C8 (4.6 mm×15 cm, 3.5 μm) or equivalent column    -   Injection amount: 20 μL    -   Flow rate: 1.0 mL/minute    -   Column temperature: 35° C.    -   Temperature for liquid detection: Constant temperature near 2˜8°        C.    -   Mobile phase A—Mixture liquid of acetate buffer¹⁾:acetonitrile        (97:3)    -   Mobile phase B—Mixture liquid of acetate buffer¹⁾:acetonitrile        (87.5:12.5)

TABLE 4 Time Mobile phase Mobile phase (minutes) A (%) B (%) 0 100 0 400 100 45 0 100 47 100 0 55 100 0 *¹⁾Acetate buffer (0.03 mol/L, pH 5.5):Liquid obtained by adding 3.4 mL of acetic acid per 2 liter of waterfollowed by thorough mixing and adjustment of pH to pH 5.5 by using 50%sodium hydroxide.

Test Example 1 Accelerated Stability Test (6 Month Stability Evaluationat 40° C., 75% RH)

As described in the above, the results of carrying out for 6 months anaccelerated stability test (40° C., 75% RH) for the composition obtainedfrom Examples 1 to 6 and Comparative examples 1 to 10 are expressed inTables 5 to 7. As for the evaluation standard of the stability test,maintaining “colorless” to “light pale yellow” was set as a standard forthe characteristics, and total impurities of 1.5% or less and individualimpurity of 0.24% or less were set as a standard, which are the same asthe management standards for impurity previously known to be generatedby oxidation.

TABLE 5 Monothio- glycerol concentration Total Individual (mg/mL) TimeCharacteristics impurities (%) impurity (%) Example 1 0.50 Initial stateColorless 0.04 0.04 4 Weeks Colorless 0.25 0.10 3 Months Colorless 0.380.14 6 Months Colorless 0.46 0.15 Example 2 0.75 Initial state Colorless0.08 0.07 4 Weeks Colorless 0.14 0.09 3 Months Colorless 0.20 0.11 6Months Colorless 0.26 0.13 Example 3 1.00 Initial state Colorless 0.080.07 4 Weeks Colorless 0.09 0.08 3 Months Colorless 0.18 0.10 6 MonthsColorless 0.24 0.11 Example 4 1.25 Initial state Colorless 0.11 0.09 4Weeks Colorless 0.12 0.10 3 Months Colorless 0.18 0.12 6 Months Lightpale yellow 0.36 0.23 Comparative 1.50 Initial state Colorless 0.09 0.08example 1 4 Weeks Light pale yellow 0.61 0.20 3 Months Light yellow 0.950.36 6 Months Light yellow 1.21 0.52 Comparative 0.10 Initial stateColorless 0.05 0.05 example 2 4 Weeks Light pale yellow 0.56 0.13 3Months Light yellow 0.71 0.24 6 Months Light yellow 0.94 0.30

As it can be confirmed from the results of Table 5 above, whenmonothioglycerol is contained at 0.50 to 1.25 mg/mL approximately as ananti-oxidant, the individual impurity (standard: 0.24% or less) andtotal impurities (standard: 1.5% or less) were maintained at thestandard level or lower during 6-month period of the acceleratedstability test, exhibiting excellent stability (Examples 1 to 4). On theother hand, when monothioglycerol concentration is within a range of0.10 mg/mL (Comparative example 2) or less, or 1.50 mg/mL (Comparativeexample 1) or more, it was found that the individual impurity exceedsthe standard with a change in the characteristics (i.e., discoloration).

TABLE 6 Oxygen Indi- concentration Total vidual in headspace Charact-impurities impurity of vial (%) Time eristics (%) (%) Example 5 0.5Initial state Colorless 0.08 0.07 4 Weeks Colorless 0.14 0.09 3 MonthsColorless 0.22 0.10 6 Months Colorless 0.26 0.11 Example 3 1.0 Initialstate Colorless 0.08 0.07 4 Weeks Colorless 0.09 0.08 3 Months Colorless0.18 0.10 6 Months Colorless 0.24 0.11 Example 6 1.5 Initial stateColorless 0.09 0.08 4 Weeks Colorless 0.14 0.09 3 Months Colorless 0.200.11 6 Months Colorless 0.26 0.12 Comparative 2.0 Initial stateColorless 0.09 0.08 example 3 4 Weeks Light pale 0.46 0.20 yellow 3Months Light 0.81 0.63 yellow 6 Months Yellow 1.10 0.92 Comparative 5.0Initial state Colorless 0.10 0.09 example 4 4 Weeks Yellow 2.32 1.32 3Months Yellow 3.08 1.59 6 Months Yellow 3.56 1.82

As it can be confirmed from the results of Table 6 above, when theoxygen concentration in the headspace of the vial is 1.5% v/v or lower,the individual impurity (standard: 0.24% or less) and total impurities(standard: 1.5% or less) were maintained at the standard level or lowerduring 6-month period of the accelerated stability test, exhibitingexcellent stability (Examples 3, 5 and 6). On the other hand, when theoxygen concentration in the headspace of the vial is higher than theaforementioned concentration (Comparative examples 3 and 4), it wasfound that the impurities exceed greatly the standard with a change inthe characteristics (i.e., discoloration).

TABLE 7 Indi- Total vidual Charact- impurities impurity Anti-oxidantTime eristics (%) (%) Comparative — Initial state Colorless 0.08 0.07example 5 4 Weeks Yellow 1.46 0.80 3 Months Deep — — yellow ComparativeL-Cysteine Initial state Colorless 0.09 0.08 example 6 4 Weeks Yellow0.80 0.46 3 Months Deep — — yellow Comparative Thioglycolic Initialstate Colorless 0.10 0.08 example 7 acid 4 Weeks Yellow 1.34 0.39 3Months Deep — — yellow Comparative EDTA Initial state Colorless 1.200.53 example 8 4 Weeks Yellow 3.02 1.38 3 Months Deep — — yellowComparative Sodium Initial state Colorless 0.09 0.08 example 9 sulfite 4Weeks Yellow 1.70 0.56 3 Months Deep — — yellow Comparative SodiumInitial state Colorless 0.09 0.07 example 10 sulfide 4 Weeks Yellow 1.460.48 3 Months Deep — — yellow

As it can be confirmed from the results of Table 7 above, when ananti-oxidant is not contained or a conventionally used commonanti-oxidant is contained, a change in the characteristics (i.e.,discoloration) is shown or the amount of individual impurity and totalimpurities exceed the standard after 1 month of the acceleratedstability test. Furthermore, just 3 months after the storage,discoloration into deep yellow color was shown, and thus no furtherobservation was carried out thereafter. Namely, it was confirmed thatthe use of monothioglycerol for producing a pemetrexed-containingcomposition solution for injection yields significantly higher stabilitycompared to a case of using L-cysteine, sodium sulfite, sodium sulfide,or the like.

Test Example 2 Severe Stability Test (6 Month Stability Evaluation at60° C.)

As described in the above, the results of carrying out for 6 months asevere stability test (60° C.) for the composition obtained fromExamples 1 to 6 and Comparative examples 1 to 4 are expressed in Tables8 and 9. As for the evaluation standard of the stability test,maintaining “colorless” to “light pale yellow” was set as the standardfor characteristics, and total impurities of 1.5% or less and individualimpurity of 0.24% or less were set as the standard, which are the sameas the management standards for impurity previously known to begenerated by oxidation.

TABLE 8 Monothio- Indi- glycerol Total vidual concentration Charact-impurities impurity (mg/mL) Time eristics (%) (%) Example 1 0.50 Initialstate Colorless 0.04 0.04 4 Weeks Colorless 0.26 0.12 3 Months Colorless0.48 0.13 6 Months Colorless 0.51 0.13 Example 2 0.75 Initial stateColorless 0.08 0.07 4 Weeks Colorless 0.24 0.09 3 Months Colorless 0.420.15 6 Months Colorless 0.81 0.20 Example 3 1.00 Initial state Colorless0.08 0.07 4 Weeks Colorless 0.34 0.13 3 Months Colorless 0.65 0.17 6Months Colorless 0.72 0.19 Example 4 1.25 Initial state Colorless 0.110.09 4 Weeks Colorless 0.10 0.09 3 Months Colorless 0.15 0.12 6 MonthsLight pale 1.08 0.23 yellow Comparative 1.50 Initial state Colorless0.09 0.08 example 1 4 Weeks Yellow 1.49 0.34 3 Months Yellow 2.24 1.78 6Months Deep — — yellow Comparative 0.10 Initial state Colorless 0.050.05 example 2 4 Weeks Light 0.74 0.13 yellow 3 Months Yellow 1.68 0.326 Months Yellow 1.97 0.68

As it can be confirmed from the results of Table 8 above, whenmonothioglycerol is contained at 0.50 to 1.25 mg/mL approximately as ananti-oxidant, the individual impurity (standard: 0.24% or less) andtotal impurities (standard: 1.5% or less) were maintained at thestandard level or lower during 6-month period of the severe stabilitytest, exhibiting excellent stability (Examples 1 to 4). On the otherhand, when monothioglycerol concentration is within a range of 0.10mg/mL (Comparative example 2) or less, or 1.50 mg/mL (Comparativeexample 1) or more, discoloration into deep yellow color was shown 6months after the storage, and thus the measurement of the amount ofimpurities was no longer carried out.

TABLE 9 Oxygen Indi- concentration Total vidual in headspace Charact-impurities impurity of vial (%) Time eristics (%) (%) Example 5 0.5Initial state Colorless 0.08 0.07 4 Weeks Colorless 0.19 0.11 3 MonthsColorless 0.21 0.14 6 Months Colorless 0.31 0.17 Example 3 1.0 Initialstate Colorless 0.08 0.07 4 Weeks Colorless 0.34 0.13 3 Months Colorless0.65 0.17 6 Months Colorless 0.72 0.19 Example 6 1.5 Initial stateColorless 0.09 0.08 4 Weeks Colorless 0.19 0.11 3 Months Colorless 0.330.16 6 Months Colorless 1.08 0.18 Comparative 2.0 Initial stateColorless 0.09 0.08 example 3 4 Weeks Light 0.79 0.25 yellow 3 MonthsYellow 1.25 0.84 6 Months Deep — — yellow Comparative 5.0 Initial stateColorless 0.10 0.09 example 4 4 Weeks Yellow 3.17 1.79 3 Months Deep — —yellow 6 Months Deep — — yellow

As it can be confirmed from the results of Table 9 above, when theoxygen concentration in the headspace of the vial is 1.5% v/v or less,the individual impurity (standard: 0.24% or less) and total impurities(standard: 1.5% or less) were maintained at the standard level or lowerduring 6-month period of the severe period of 6 months, exhibitingexcellent stability (Examples 3, 5, and 6). On the other hand, when theoxygen concentration in the headspace of the vial is higher than theaforementioned concentration (Comparative examples 3 and 4), a change inthe characteristics (i.e., discoloration) was shown and the impuritiesare significantly higher than the 3-month severe storage standard.

In a case in which the oxygen concentration in the headspace of the vialis 2.0% (Comparative example 3) or 5.0% (Comparative example 4),discoloration into deep yellow color was shown 6 months and 3 monthsafter the storage, respectively, and thus no further measurement of theamount of impurities was carried out. It was accordingly recognizedthat, when monothioglycerol as an anti-oxidant is contained within aspecific concentration range and oxygen concentration in the headspaceof the vial is kept at 1.5% or less for producing a solution forinjection which contains pemetrexed and a salt thereof, a formulationhaving remarkably improved stability not only at general temperatureconditions but also at severe storage condition of 60° C. can beobtained.

1. A composition comprising pemetrexed or a pharmaceutically acceptablesalt thereof as an active ingredient and 0.50 mg/mL to 1.25 mg/mL ofmonothioglycerol as an antioxidant.
 2. The composition according toclaim 1, wherein monothioglycerol as an antioxidant is comprised at theamount of 0.50 to 1.00 mg/mL.
 3. The composition according to claim 2,wherein monothioglycerol as an antioxidant is comprised at the amount of0.50 to 0.80 mg/mL.
 4. A composition comprising pemetrexed or apharmaceutically acceptable salt thereof as an active ingredient andmonothioglycerol as an antioxidant in which the active ingredient andmonothioglycerol are comprised at ratio of about 20:1 to 50:1.
 5. Thecomposition according to claim 4, wherein the active ingredient andmonothioglycerol are comprised at ratio of about 25:1 to 50:1.
 6. Thecomposition according to claim 1, wherein the active ingredient iscomprised at the amount of 20 to 100 mg/mL.
 7. The composition accordingto claim 1, wherein the active ingredient is pemetrexed disodium 2.5hydrate.
 8. The composition according to claim 1, wherein the activeingredient is pemetrexed disodium 7 hydrate.
 9. A liquid preparationcomprising the composition of claim 1 in the form of a solution.
 10. Theliquid preparation according to claim 9, wherein dissolved oxygen amountin the solution is not more than 0.5 ppm.
 11. The liquid preparationaccording to claim 9, wherein pH of the solution is 6.0 to 8.0.
 12. Theliquid preparation according to claim 9, wherein the liquid preparationis packaged in a sealed container, and concentration of oxygen containedin the headspace gas of the sealed container is not more than 1.5% v/v.13. The liquid preparation according to claim 12, wherein theconcentration of oxygen contained in the headspace gas of the sealedcontainer is not more than 1.0% v/v.
 14. The liquid preparationaccording to claim 9, wherein an individual impurity is maintained at0.24% or less and total impurities are maintained at 1.50% or less for 3months or longer at 40° C. and relative humidity of 75%.
 15. The liquidpreparation according to claim 9, wherein an individual impurity ismaintained at 0.24% or less and total impurities are maintained at 1.50%or less for 3 months or longer at 60° C.
 16. A liquid preparationproduced by comprising steps of: dissolving pemetrexed or apharmaceutically acceptable salt thereof and 0.50 to 1.25 mg/mL ofmonothioglycerol in water for injection; and controlling oxygenconcentration in headspace gas of a sealed container to 1.5% v/v orless.
 17. The composition according to claim 4, wherein the activeingredient is pemetrexed disodium 2.5 hydrate.
 18. The compositionaccording to claim 4, wherein the active ingredient is pemetrexeddisodium 7 hydrate.
 19. A liquid preparation comprising the compositionof claim 4 in the form of a solution.
 20. The liquid preparationaccording to claim 19, wherein dissolved oxygen amount in the solutionis not more than 0.5 ppm.
 21. The liquid preparation according to claim19, wherein pH of the solution is 6.0 to 8.0.
 22. The liquid preparationaccording to claim 19, wherein the liquid preparation is packaged in asealed container, and concentration of oxygen contained in the headspacegas of the sealed container is not more than 1.5% v/v.
 23. The liquidpreparation according to claim 22, wherein the concentration of oxygencontained in the headspace gas of the sealed container is not more than1.0% v/v.
 24. The liquid preparation according to claim 19, wherein anindividual impurity is maintained at 0.24% or less and total impuritiesare maintained at 1.50% or less for 3 months or longer at 40° C. andrelative humidity of 75%.
 25. The liquid preparation according to claim19, wherein an individual impurity is maintained at 0.24% or less andtotal impurities are maintained at 1.50% or less for 3 months or longerat 60° C.